Hydrogen peroxide formation by Nox4 limits
malignant transformation
Valeska Helfinger1, Florian Freiherr von Gall1, Nina Henke2, Michael M. Kunze2, Tobias
Schmid2, Juliana Heidler3, Ilka Wittig3,9, Heinfried H. Radeke4, Viola Marschall6, Karen
Anderson7, Ajay M. Shah8, Simone Fulda6, Bernhard Brüne2, Ralf P. Brandes1, Katrin
Schröder1
1: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt am Main, Germany
2: Institute for Biochemistry I/ Pathobiochemistry, Goethe-University, Frankfurt am Main,
Germany
3: Functional Proteomics, SFB 815 Core Unit, Goethe-University, Frankfurt am Main,
Germany
4: Pharmazentrum Frankfurt, Goethe-University, Frankfurt am Main, Germany
5: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt,
Germany; German Cancer Consortium (DKTK), Heidelberg, Germany ,German Cancer
Research Center (DKFZ), Heidelberg, Germany
6: The Babraham Institute, Babraham Research Campus, Cambridge, UK
7: King’s College London British Heart Foundation Centre, Cardiovascular Division, London,
UK
8: Cluster of Excellence “Macromolecular Complexes”, Goethe University, Frankfurt am
Main, Germany
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Conflict of interest disclosure:
None of the authors has any conflict of interest to disclose
*Author for correspondence:
Katrin Schröder
Institut für Kardiovaskuläre Physiologie
Fachbereich Medizin der Goethe-Universität
Theodor-Stern Kai 7
60596 Frankfurt
Germany
Email: [email protected]
Running Head: Nox4 averts cancer
Keywords: Nox4, cancer, genomic instability
Precis
By oxidizing AKT and keeping PP2A in the cytosol, the NADPH oxidase Nox4 allows proper
DNA damage repair and averts cancer development.
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Abstract
Reactive oxygen species (ROS) can cause cellular damage and are thought to promote
cancer-development. Nevertheless, under physiological conditions, all cells constantly
produce ROS, either as chemical by-products or for signaling purpose. During differentiation
cells induce the NADPH oxidase Nox4, which constitutively produces low amounts of H2O2.
We infer that this constitutive H2O2 is unlikely to be carcinogenic and may rather maintain
basal activity of cellular surveillance systems.
Utilizing two different murine tumor models we demonstrate that Nox4 prevents malignant
transformation and facilitated the recognition of DNA-damage. Upon DNA-damage repair is
initiated as consequence of phosphorylation of H2AX (γH2AX). Repair only occurs if nuclear
activity of the γH2AX-dephosphorylating phosphatase PP2A is kept sufficiently low, a task
fulfilled by Nox4: Nox4 continuously oxidizes AKT, and once oxidized AKT captures PP2A in
the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation
of γH2AX. Simultaneously the proportion of active, phosphorylated AKT is increased. Thus,
DNA-damage is not recognized and the increase in AKT activity promotes proliferation. The
combination of both events resulted in genomic instability and tumor initiation.
With the identification of the first cancer-protective source of reactive oxygen species, Nox4,
the paradigm of reactive-oxygen species-induced initiation of malignancies should be
revised. (200 words)
Significance
The stereotype of ROS produced by NADPH oxidases as cause of malignant diseases
persists generalized since decades. We demonstrate that the NADPH oxidase Nox4, as
constitutive source of ROS, prevents malignant transformation and that its pharmacological
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inhibition as currently aspired by several companies will potentially increase the risk of
malignant cell transformation and eventually tumor formation.
Introduction
The notion that reactive oxygen species (ROS) are necessary harmful and are the cause of
aging and all main diseases is very prevalent. Nevertheless, the prospective randomized
clinical trials on antioxidant supplementation have failed to provide protection and in some
studies antioxidants even increased the risk of negative outcome (1, 2). Recently, animal
experiments demonstrated that some major antioxidants of clinical use, N-acetylcysteine and
vitamin E, accelerate spontaneous lung cancer development in mice carrying mutations in K-
Ras and B-Raf (3).
A potential explanation for these findings is that ROS are not just a representation of
environmental stress or inflammation but that basically all cells produce ROS for signaling
purpose. Such kinds of ROS are generated by the Nox family of NADPH oxidases in a highly
controlled fashion (4). Nox-dependent ROS-production in general occurs in response to
cellular stimulation through increases in intracellular calcium or activation of Rac and protein
kinase C (5). There is however one exception, the NADPH oxidases Nox4. This enzyme
constitutively produces low amounts of H2O2 and thus its output is controlled by the
expression level of the enzyme. Hypoxia and TGFβ are potent inducers of Nox4 expression
(6, 7). Therefore, increased Nox4 expression in disease conditions can be considered a
marker of cellular stress.
Interestingly, some tumor suppressor genes, particularly Nrf2, are induced by ROS and
facilitate the antioxidant response and cellular protection (8). In previous work we reported
that Nox4 maintains the expression of Nrf2 (9). Moreover, Nox4-/- mice exhibit a hyper-
inflammatory response in the vasculature which results in cardiovascular dysfunction and
accelerated arteriosclerosis (9–13). We and others also found Nox4 to be induced in the
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course of differentiation and to be essentially involved in the differentiation process of
mesenchymal cells (14–16). As de-differentiation and inflammation are well established pre-
requisites for malignant transformation we hypothesized that the knockout of Nox4 increases
the risk of malignancy development.
Using two different inflammation driven cancer models as well as cell culture studies, we
here demonstrate that endogenous Nox4 maintains genomic stability and attenuates
malignant transformation. Nox4 mediates its effect through oxidation of AKT, which
subsequently promotes cytosolic PP2A sequestration and PP2A-mediated AKT
dephosphorylation. Cytosolic trapping of AKT leads to attenuated nuclear PP2A deposition
which facilitates DNA damage detection.
Materials and Methods
Materials
The following chemicals were used: 3-methylcholanthrene (MCA), azoxymethane (AOM),
NaCl, NH4Cl, NaHCO3 Hank’s BSS without Ca2+ and Mg2+ and Trypsin-EDTA solution
(T3924) from Sigma-Aldrich (Munich, Germany), Dextran sulphate sodium (DSS)
(#16011080; MP Biomedicals, Santa Ana , USA), Dulbecco’s PBS (Gibco lifetechnologies,
Carlsbad, CA, USA), Hank’s buffer, SYBR green and Na-EDTA from Applichem (Darmstadt,
Germany), TRIS (Carl Roth) and fibronectin (Corning Incorporated, Tewksbury, MA, USA).
Collagenase Type 2 was purchased from Worthington (Lakewood, NJ, USA). The PI3-Kinase
inhibitor Ly294002 (#BML-ST420) was acquired from Enzo Life Science (Lörrach, Germany).
The following antibodies were used: Anti-pH3 (06-570), anti-PP2A and anti-phospho-Histone
H2A.X (Ser139) from Millipore (Darmstadt, Germany), anti- AKT, anti-phospho-AKT
(Ser473), p-p53 (Ser15), ATM, HA and PTEN, Erk, p-Erk, AKT sepharose bead conjugate
and IgG mouse sepharose bead conjugate from CellSignaling (Danvers, MA, USA), anti-p53
(FL-393), anti-p110, NQO1, p-ATM (Ser1981), GFP and anti-Topoisomerase 1 (C-15) from
Santa Cruz (Dallas, TX, USA), anti- β-actin (AC-15) from Sigma-Aldrich (Munich, Germany)
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and anti-p85 from BD transduction laboratories (San Jose, CA, USA). Fluorogenic substrates
for proteasome activity were purchased from Boston Biochem (Minneapolis, USA). Human
recombinant AKT1 (# ab116412) was purchased from Abcam (Cambridge, United Kingdom).
Animals and animal procedures
All animal experiments were approved by the local governmental authorities (approval
number: F28/27 and F28/46) and were performed in accordance with the animal protection
guidelines. C57Bl/6J and Nox2y/- mice were purchased from Charles River (Deisenhofen,
Germany). Nox4 -/- mice were generated by targeted deletion of the translation initiation site
and of exons 1 and 2 of the Nox4 gene (9), backcrossed into C57Bl/6J for more than 10
generations. Tamoxifen-inducible Nox4-/- mice (Nox4flox/flox-ERT2-Cre+/0 mice) were
produced by crossing Nox4flox/flox mice (backcrossed more than 10 generations into
C57Bl/6J) with Cre-ERT2+/0 mice (9). Genetic deletion of Nox4 in Nox4flox/flox-ERT2-
Cre+/0 mice was achieved by oral tamoxifen administration in the chow (LASCRdiet
CreActive TAM400, LASvendi, Soest, Germany) on 10 consecutive days. Both Cre positive
as well as the Cre negative littermates received tamoxifen. Nox1y/- mice, kindly provided by
Karl-Heinz Krause and previously characterized were used for the same experiments (18).
Mice were housed in a specified pathogen-free facility with 12/12 hours day and night cycle
and free access to water and chow every time.
To induce fibro sarcomas the chemical carcinogen MCA was injected subcutaneously into
the right flank of the mice. In case tumors reached a diameter of 1.5 cm or 150 days after
MCA-injection mice were sacrificed and if present the tumor tissue was used for cell
isolation, immuno-histological and biochemical analysis.
To analyze the short term effect of MCA-injection mice were sacrificed 20 days after MCA
injection and skin of injection site was used for histological and biochemical analysis.
Colon carcinomas were induced as follows: A single dose of 10mg/kg body weight AOM was
injected intraperitoneally. After 1 week mice were treated for 1 week with 2% DSS in the
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drinking water followed by two weeks with usual drinking water. This procedure was repeated
2 additional times. Two weeks after the third DSS cycle mice were sacrificed and the colon
was used for further analysis.
Cell culture
Fibro sarcoma cells were isolated using the tumor dissociation kit for mouse and the gentle
MACS Dissociator from Miltenyi Biotec (Bergisch-Gladbach, Germany), following the
manufactures instructions. Shortly, tumor tissue was homogenized enzymatically,
erythrocytes were lysed and eventually cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) + glutaMAX (Gibco, life technologies; Carlsbad, CA, USA) supplemented
with 5% fetal calf serum (FCS) and 1% penicillin (50 U/ml) and streptomycin (50 µg/ml) in a
humidified atmosphere of 5% CO2 at 37°C. Erythrocyte depletion buffer: 155 mM NH4Cl, 10
nM NaHCO3 and 100nM EDTA in double distilled water, pH=7.4.
For isolation of dermal skin fibroblasts from mice 1 cm2 piece of skin was cut into small
pieces, transferred into 2.5 ml Collagenase solution (1000 U/ml in Hanks without Ca2+/Mg2+)
and incubated on shaker for 105 minutes at 37°C. Afterwards the solution was re-suspended
and transferred through a 70 µm nylon filter. Cells were centrifuged and placed on a
fibronectin-coated dish. The primary fibroblasts are cultured in DMEM/F-12 (Gibco, life
technologies) supplemented with 10% FCS and 1% Pen/Strep in a humidified atmosphere of
5% CO2 at 37°C. Murine lung endothelial cells were isolated and cultured as described by
Schröder et al. (9).
Site-directed mutagenesis and transfection of plasmids
The QuickChange II XL Site-Directed Mutagenesis Kit (#200521, Agilent) was used to make
point mutations of AKT1/2/3. Corresponding primers were designed with QuickChange
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Primer Design Program. Plasmids for mouse AKT1 (#39531), AKT2 (#64832) and AKT3
(#27293) from Addgene were used for converting the two cysteine residues of the specific
AKT into serine to create mutants. The following primers were used for the introduction of
single amino acid mutations: AKT1 C296S 5’-ATC CCC TCC TTG CTC AGC CCG AAG
TCC-3’ and 5’-GGA CTT CGG GCT GAG CAA GGA GGG GAT-3’; AKT1 C310S 5’-CTC
CGG CGT TCC GCT GAA TGT CTT CAT AGT GGC-3’ and 5’- GCC ACT ATG AAG ACA
TTC AGC GGA ACG CCG GAG-3’; AKT2 C297S 5’- CTG ATG CCC TCT TTG CTC AAG
CCA AAG TCA GTG-3’ and 5’-CAC TGA CTT TGG CTT GAG CAA AGA GGG CAT CAG-3’;
AKT2 C311S 5’- TAC TCC GGG GTA CCA CTG AAG GTT TTC ATG GTG-3’ and 5’- CAC
CAT GAA AAC CTT CAG TGG TAC CCC GGA GTA-3’; AKT3 C293 S 5’- CTG TGA TCC
CTT CTT TGC TAA GCC CAA AAT CCG TAA TTT TT-3’ and 5’-AAA AAT TAC GGA TTT
TGG GCT TAG CAA AGA AGG GAT CAC AG-3’; AKT3 C307S 5’- GTA CTC TGG TGT
GCC ACT GAA TGT CTT CAT GGT AG-3’ and 5’- TAC CCA TGA AGA CAT TCA GTG GCA
CAC CAG AGT AC-3’. The double mutants (C296/310, C297/311, C293/307) were
constructed using the single mutants as DNA template and primers for
C310S/C311S/C307S. The experiments were performed according to the manufacturers
protocol and mutations were verified by sequencing. AKT1 S473A/T308A mutant was a gift
from Itamar Goren and AKT K179M from Beate Fisslthaler. Transfection of basal AKT
plasmids and mutants was performed with Lipofectamine 3000 (#L3000-015) according to
the manufacturer’s instructions.
Proximity ligation assay (PLA)
Analysis was performed as described in the manufacturer’s protocol (Duolink II
Fluorescence, OLink, Upsalla, Sweden). Briefly, isolated fibro sarcoma cells were fixed in
phosphate buffered formaldehyde solution (4%), permeabilized with Triton X-100 (0.2%),
blocked with serum albumin solution (3%) in phosphate buffered saline and incubated
overnight with appropriate antibodies. After washing, samples were incubated with respective
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PLA-probes for 1 hour, washed and ligated for 30 min, both at 37°C. An additional washing
step followed and amplification with polymerase was performed for 100 min. Images were
obtained by confocal microscopy with LSM 510 (Zeiss, Jena, Germany). Fiji software was
used for quantification of single dots per cell.
Protein and Western Blot analysis
For whole cell protein isolation, cells were lysed in a buffer containing 20mM TRIS/cl pH 7.5,
150 nM NaCl, 10mM NaPPi, 20 nM NaF, 1% Triton, 10nM Okadaic acid (OA), 2mM
Orthovanadat (OV), protein-inhibitor mix (PIM) and 40 µg/ml phenylmethylsulfonylfluorid
(PMSF).
For separation of nucleus and cytosol, the cells were lysed in buffer A (10 nM HEPES pH
7.9, 10 nM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet, 10 mM DTT, protein-inhibitor
mix (PIM), 40 µg/ml phenylmethylsulfonylfluorid (PMSF). Cells were centrifuged to gain the
cytosol containing supernatant. The pellet was further lysed with buffer B (20 mM HEPES pH
7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM DTT, protein-inhibitor mix (PIM), 40
µg/ml phenylmethylsulfonylfluorid (PMSF)) to obtain the nuclear extract. Bradford assay was
used to determine the protein amount (19). Samples were cooked in sample buffer and were
transferred on SDS-PAGE followed by Western Blotting. Identical amounts of protein from
nuclear and cytosolic fractions were loaded. Analysis was performed with an infrared-based
detection system using fluorescent-dye-conjugated secondary antibodies from LI-COR
biosciences.
Phosphatase activity was analyzed according to the manufacturer’s suggestion. Briefly, 50
mM pNPP were incubated with lysates of cytosolic or nuclear fractions prepared as
described above. After 5-10 minutes the reaction was stopped by addition of NaOH and the
amount of the product of the phosphatase reaction, p-nitrophenol, was determined by
reading the absorbance at 405 nm in a microplate reader (Tecan Infinite 200 Pro).
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BIAM Switch Assay was performed to determine the oxidation state of AKT. Briefly, cells
were blocked with n-ethylmaleimide (NEM) and scratched in TCA. To block the free thiols
cell pellets were resuspended in NEM-denaturing buffer (containing Tris-HCL pH 8.5, Urea,
EDTA and SDS) subsequently followed by acetone precipitation. Reduction of oxidized thiols
was performed with DTT-denaturing buffer and labeling of those with biotin-
polyethyleneoxide-iodoacetamide-denaturing buffer followed by acetone precipitation and
triton lysis. Lysates were used for IP with streptavidin-agarose beads and blotted for AKT.
mRNA isolation and RT-qPCR
Total mRNA from cells and frozen homogenized tissue was isolated with a RNA-Mini-kit
(Bio&Sell, Feucht, Germany) according to the manufacturers protocol. Random hexamer
primers (Promega, Madison,WI, USA) and Superscript III Reverse Transcriptase (Invitrogen,
Darmstadt, Germany) were used for cDNA synthesis. Semi-quantitative real-time PCR was
performed with Mx3000P qPCR cycler (Agilent Technologie, Santa Clara, CA, USA) using
PCR Eva Green qPCR Mix with ROX (Bio&Sell, Feucht, Germany) with appropriate primers.
Relative expression of target genes were normalized to eukaryotic translation elongation
factor 2 (EF2), analyzed by delta-delta-Ct method and given as percentage compared to
control experiments. Primer sequences for murine Nox4 were forward 5’-
TGTTGGGCCTAGGATTGTGTT-3’ and reverse 5’-AGGGACCTTCTGTGATCCTCG-3’. For
p53 forward 5’-AGACCGCCGTACAGAAGAAG3’ and reverse 5’-
TTCAGCTCCCGGAACATCTC-3’, Cyp1A1 forward 5‘-GGCCACTTTGACCCTTACAA-3’ and
reverse 5‘-CAGGTAACGGAGGACAGGAA-3’ -3’, for Cyp1B1 forward 5‘-
TTCTCCAGCTTTTTGCCTGT-3’ and reverse 5‘-TAATGAAGCCGTCCTTGTCC-3’ and ATM
forward 5’-ATGCCAGTCTTTTCAGGGTG-3‘ and reverse 5‘-TCAGAAGCTGGGAGTGCTTC-
3’.
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DNA-damage detection (Comet- and Nicoletti-Assay)
A suspension of 1*106 cells/ml was mixes 1:10 with 5% low melting agarose and subjected
onto slides coated with 1.5% normal melting agarose. Lysis of the cells was performed for 2
hours at 4°C in lysis buffer (2.5 M NaCl, 10 mM TRIS, 100 mM EDTA, pH=10., 1% Triton X-
100 and 10% SDS in double distilled water). Lysis was followed by a 20 minutes incubation
of the slides on ice with the alkaline electrophoresis buffer (300 mM NaOH and 0.5 M EDTA)
with subsequent electrophoresis at 25 V for 20 minutes. Slides were washed three times with
PBS and were stained with SYBR green. Pictures were taken with a confocal microscope
LSM 510 Meta and quantification was done manually by three independent investigators
determining the ratio of cell number/cells with comets.
Additionally DNA fragmentation was determined by analysis of propidium iodide (PI)-stained
nuclei using flow cytometry as described previously (20). Cells were lysed and stained for 2 h
in a solution of 0.1% tri-sodium citrate dehydrate and 0.1% Triton X-100 containing 50 µg/ml
propidium iodide (Sigma, Deisenhofen, Germany) and analyzed by flow cytometry
(FACSCanto II, BD Biosciences, Heidelberg, Germany).
Immunohistochemistry
Tissue was fixed in 4% paraformaldehyde overnight, dehydrated in ascending ethanol-series
and then embedded in paraffin. Paraffin blocks were sliced and slides were dewaxed for
further staining in descending ethanol-series from 100% to 70%. For antigen retrieval slides
were cooked 10 min in citrate 1 x TRS buffer (Dako). After cooling down staining procedure
started using the Dako CSA system in combination with Biotin blocking System (Dako) for
3,3’ diaminobenzidine staining. First, slides were block with Avidin and Biotin for 20 minutes
with the biotin blocking system from Dako, followed by peroxidase and protein blocking from
the Dako staining kit. In between washing steps were performed with TBST. After the protein
block first antibody was applied on the slides overnight at 4°C. The next day slides were
incubated with biotinylated secondary antibody, amplification steps were performed as
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described in the manual and diaminobenzidine was used to generate the signal.
Counterstaining was performed with hematoxylin.
Polysomal Fractionation
Isolated fibro sarcoma cells were seeded in a 15 cm dish one day prior to harvesting.
Polysome analysis was performed as described previously (21). Briefly, after incubation with
100 µg/ml cycloheximide for 10 min at 37°C cells were scraped and centrifuged. The
supernatant was discarded, pellet washed, centrifuged again and lysed in 750 µl polysome
buffer (140 mM KCl, 20 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.5% NP40, 0.5 mg/mL heparin,
1 mM DTT, 100 U/mL RNasin (Promega), 100 μg/mL CHX). After pelleting for 5 min, 16000
rpm at 4°C and transferring the supernatant into a fresh tube, 600 µl of cytoplasmic lysate
was layered onto a 10-50% continuous sucrose gradient. The gradient was centrifuged at
35000 rpm for 2 h without brake, and the gradients were collected in 1-mLfractions using a
Gradient Station (Biocomp). Absorbance was measured at 254 nm. RNA was precipitated by
addition of sodium acetate (3 M) and isopropanol. RNA was further purified using the
Nucleospin RNA Kit (Macherey-Nagel) according to the manufacturer's manual and analyzed
by RT-qPCR.
Mass spectrometry measurements of inositol lipids
Mass spectrometry was used to measure inositol lipid levels essentially as described
previously (22) using a QTRAP 4000 (AB Sciex) mass spectrometer and employing the lipid
extraction and derivatization method described for cultured cells (cells isolated from tumors)
and whole tissue (tumor tissue), with the modification that initial samples were probe
sonicated for 5” (using a microtip) prior to extraction and that final samples were dried in a
speedvac concentrator rather than under N2. Measurements were conducted on 1 x106
isolated fibro sarcoma cells or 1mg wet weight tumor tissue per sample. C16:0/C17:0 PI
(100ng) and PIP3 (10ng) internal standards (ISDs) were added to each sample prior to
extraction. Integrated area of lipid species peaks were corrected for recovery against ISD
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area (giving a response ratio for each lipid) and data expressed as C18:0 C20:4 PIP3
response ratio normalized to C18:0 C20:4 PI response ratio to account for cell input
variation. Both endogenous PIP3 and PI were corrected to their own internal standard, and
then the one measurement is divided by the other, to get the best estimate of true PIP3/PI.
Sample preparation and mass spectrometry
2.5 µM recombinant AKT1 (Abcam) were reduced in 100 µM DTT and treated with 300µM
H2O2 for 30 min. Proteins were directly digested overnight with trypsin (sequencing grade,
Promega) and analyzed by liquid chromatography / mass spectrometry (LC/MS).
Fibro sarcoma cells of wildtype and Nox4-deficient mice were lysed in buffer A. Lysates were
used to trap AKT1-3 including interacting proteins using a sepharose beads immobilized AKT
antibody and IgG (CellSignaling) as negative control. Beads were washed in PBS,
resuspended in 50 µl 8 M Urea, 50 mM Tris/HCl, pH 8.5, and incubated at 22°C for 30 min.
Thiols were alkylated with 40 mM chloroacetamid and samples were diluted with 25 mM
Tris/HCl, pH 8.5, 10% acetonitrile to obtain a final urea concentration of 2 M. Proteins were
digested with 1 µg Trypsin/LysC (sequencing grade, Promega) overnight at 22°C under
gentle agitation. Digestion was stopped by adding trifluoroacetic acid to a final concentration
of 0.1 %. Peptides were loaded and purified on multi-stop-and-go tip (StageTip) containing
six C18-disks (23). Peptides were dried and resolved in 1% acetonitrile, 0.1 % formic acid
and analyzed by LC/MS.
LC/MS was performed on Thermo Scientific™ Q Exactive Plus equipped with an ultra-high
performance liquid chromatography unit (Thermo Scientific Dionex Ultimate 3000) and a
Nanospray Flex Ion-Source (Thermo Scientific). Peptides were loaded on a C18 reversed-
phase precolumn (Thermo Scientific) followed by separation on a with 2.4 µm Reprosil C18
resin (Dr. Maisch GmbH) in-house packed picotip emitter tip (diameter 100 µm, 15 cm long,
New Objectives) using an gradient from mobile phase A (4% acetonitrile, 0.1% formic acid)
to 30 % mobile phase B (80% acetonitrile, 0.1% formic acid) for 20 min (purified human
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AKT1) or 90 min (AKT1 enriched from lysate) followed by a second gradient to 60% B for 10
min or 15 min, respectively. MS data were recorded by data dependent acquisition Top10
method selecting the most abundant precursor ions in positive mode for HCD fragmentation.
The full MS scan range was 300 to 2000 m/z with resolution of 70000, and an automatic gain
control (AGC) value of 3*106 total ion counts with a maximal ion injection time of 160 ms.
MS/MS scans were recorded with a resolution of 17500, an isolation window of 2 m/z and an
AGC value set to 105 ions with a maximal ion injection time of 150 ms. Selected ions were
excluded in a time frame of 30s following fragmentation event.
Data analysis of purified human AKT1: Xcalibur raw files were analyzed by Peaks7 Studio
software for proteomics (www.bioinfor.com; Bioinformatics Solutions, Waterloo, ON,
Canada). The enzyme specificity was set to trypsin. Missed cleavages were limited to 3.
Monoisotopic precursor mass error tolerance was 5 ppm, and fragment ion tolerance was
0.05 Da. Following variable modifications were selected: of methionine (+15.99), disulfide
bridge (-2.02), half of disulfide bridge (-1.01), dioxidation on cysteines (+31.99) and
trioxidation on cysteines (+47.98). After de novo sequencing of spectra, the human reference
proteome set (download from Uniprot, April 4th, 2015; 68511 entries; www.uniprot.org) was
used to identify peptide-spectrum matches with a false discovery rate (FDR) of 1%. For
identification of crosslinked peptides by disulfide bridges, a special software StavroX
(v3.4.12) (24) was used. Disulfide bridge (-2.01565) was included and the search for di-
peptides was done using precise scoring mode. Only di-peptides with highest scores (>200)
were inspected and shown in supplementary table IW2. Disulfide bridges within the structure
of AKT1 (4EJN, (25)) were illustrated using Pymol (0.99rev9)).
Data analysis of immune-trapped AKT1-3: Xcalibur raw files were analyzed by MaxQuant
(1.5.2.8 (26)) with specificity to trypsin and tolerated missed cleavages of 2. Following
variable modifications were selected: of methionine (+15.99), carbamidomethylation on
cysteines (+57.02) and acetylation on N-terminus (+42.01). Proteins were identified using
proteome set of mouse (Uniprot, 26th June, 2015, 76086 entries) with a false discovery rate
(FDR) of 1%. Proteins were quantified using lable free quantification (LFQ) with at least one
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peptide and further analyzed by Perseus 1.5.4.1. (27). Reverse hits, known contaminants
and identified by side proteins were removed from the list. Missing LFQ values were replaced
by a background value generated from the normal distribution to enable calculation of ratios
to control. Permutation test was performed to identify interacting proteins on AKT1-3.
Statistics
All values are mean±SEM. Statistical analysis was performed by ANOVA followed by LSD
post hoc testing or by t-test, if appropriate. Tumor free survival curves were compared by
ANOVA for repeated measurements. Densitometry was performed with the odyssey-
software. A p-value of less than 0.05 was considered statistically significant.
Results
Loss of Nox4 promotes tumor development.
In order to determine the impact of Nox4 on tumor development, knockout mice were used.
As compared to WT mice, Nox4 knockout mice develop twice as many tumors in the
AOM/DSS-colon carcinoma model (Fig. 1A-C). Similar results were obtained in a second
model, fibrosarcoma-development in response to MCA. In Nox4-/- mice, as compared to WT
animals, the onset of tumor development was earlier and the developing tumors grew faster
(Fig. 1D-F). Proliferation within the both tumor models was more than twice as high in Nox4-
/- mice as compared to WT mice (Fig. 1G&H). Importantly, these effects were specific for
Nox4: Deletion of Nox1 rather had an inhibitory effect on tumor development, and deletion of
Nox2 was without effect at all (Supplemental figure 1). To substantiate the importance of
Nox4 as inhibitory enzyme for tumor development, conditional Nox4 knockout mice were
studied. Tamoxifen-induced systemic deletion of Nox4 resulted in a similar reduction of tumor
development as observed in the global knockout mice. Conditional deletion of Nox4 in
macrophages or endothelial cells, in contrast, was without effect on tumor development,
suggesting that most probably Nox4 in the transforming cells but not in the tumor stroma
mediates the protective effect (Supplemental figure 2).
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Knockdown of Nox4 results in genomic instability.
As genomic instability is an important factor promoting tumor development, cell lines from
WT and Nox4 tumors were established and comet assays as a marker for DNA
fragmentation were performed. Nox4 knockout tumor cells exhibited a doubling in the
number of comets. Importantly, this effect was a consequence of a shortage of H2O2, the
product of Nox4. When the tumor cells were treated with H2O2 (5µmol/L, 24h) the number of
comets in Nox4-/- tumor cells was reduced to that in the WT tumor cells, whereas WT tumor
cells did not respond to this low concentration of H2O2 (Fig. 1I&J). These observations
suggest that lack of Nox4-derived H2O2 may promote genomic instability. Indeed, Nicoletti
staining of lung, liver and intestine of healthy Nox4-/- mice demonstrated that DNA strand
breaks are more frequent after deletion of Nox4 even in normal tissue (Fig. 1K).
Loss of Nox4 results in attenuated recognition of DNA damage.
The increased DNA fragmentation in healthy Nox4-/- mice points towards an attenuated
recognition or DNA damage response after deletion of the ROS generator. Indeed, markers
of DNA damage like phosphorylation of histone 2AX (here termed γH2AX) were decreased in
tumors and tumor cells of Nox4-/- mice as compared to WT mice. Importantly, already in the
healthy colon of Nox4-/- mice or in fibroblasts cultured from those mice, the levels of γH2AX
were attenuated, which speaks for a general attenuation of the DNA damage response (Fig.
2A-D). Accordingly, acute exposure of fibroblasts to the carcinogen MCA increased γH2AX
less effectively in Nox4-/- cells as compared to WT cells (Fig. 2D). P53 is an important
transcription factor responding to DNA damage. In line with the γH2AX data, p53 protein and
mRNA expression was reduced in Nox4 knockout tumors and cells isolated from the tumor
as compared to WT controls (Fig. 2E-H). Importantly, treatment with H2O2, the product of
Nox4 normalized p53 mRNA expression in Nox4-deficient cells but had no effect in WT cells
(Fig. 2F). The decreased p53 expression was not a consequence of altered phosphorylation
or translation of p53 (Supplemental figure 3A-D) nor was the expression of enzymes
involved in p53 degradation, like NQO1 and MDM2 increased in Nox4-/- cells
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(Supplemental figure 3E&F). In order to study whether the reduced p53 expression is a
consequence of an inappropriate response to damage, we stimulated healthy endothelial
cells cultured from the lung of WT and Nox4-/- mice with the carcinogen MCA. Whereas p53
expression was strongly induced in cells of WT mice, the knockout mice exhibited a highly
attenuated response (Fig. 2I). This indicates that DNA damage response is attenuated after
deletion of Nox4. Importantly, expression and activity of ATM, the kinase which
phosphorylates H2AX in response to damage and initiates the DNA damage response was
not reduced in Nox4-deficient cells (Supplemental figure 4).
Nox4-deficiency enhances nuclear PP2A activity
Steady state levels of γH2AX depend on the activity of the H2AX phosphorylating ATM and
the γH2AX phosphatase. The latter task is carried out by the important serine-threonine
phosphatases of the PP2A family (28, 29) .Given that ATM expression and activity was not
altered after Nox4 knockout, we hypothesized that nuclear PP2A activity is increased in
Nox4-/- cells. Indeed, by proximity ligation assay (PLA) with a pan-C-subunit antibody of the
PP2A family an increased association of a PP2A member with γH2AX in the nucleus was
observed (Fig. 3A). Whereas the total cellular expression of PP2A was similar between WT
and Nox4-/- cells, the nuclear PP2A abundance was greatly increased in Nox4-/- cells as
judged by the abundance of the C-subunit (Fig. 3B). As a consequence, global serine-
threonine phosphatase activity was decreased in the cytosol and increased in the nucleus of
Nox4-/- cells when compared to the corresponding WT cells (Fig. 3C). In order to link these
data to PP2A activity, experiments with the pan-PP2A inhibitor okadaic acid were carried out
at a concentration of 1 nM. At this concentration, the inhibitor is thought to be very selective
for PP2A, whereas at higher concentrations, other phosphatases are blocked too. (30). While
okadaic acid (1 nM) had no significant effect on nuclear phosphatase activity in WT cells, it
reduced the phosphatase activity in Nox4-/- cells to the level observed in WT cells (Fig. 3C).
In keeping with this, okadaic acid restored the level of γH2AX in Nox4-/- cells to the level of
WT cells but had no effect on γH2AX level in WT cells (Fig. 3D). Strikingly, H2O2, the product
of Nox4 also restored γH2AX levels (Fig. 3E) and reduced the nuclear PP2A level in Nox4-/-
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cells to the level of WT cells (Fig. 3F). Collectively, these observations suggest that Nox4-
derived H2O2 facilitates accumulation of PP2A in the cytosol. In the absence of Nox4, and
thus lack of H2O2, PP2A accumulates in the nucleus where it dephosphorylates γ2HAX. The
result is an attenuated DNA damage response and genomic instability.
Nox4 retains PP2A in the cytosol where it interacts with and dephosphorylates AKT
The identification of the molecular basis of the nuclear PP2A accumulation after Nox4
knockout is a complex problem, given the heterogeneous composition of PP2A family
members and the fact that numerous mechanisms could account for the effect. Among them
are post-translational modifications of the target protein leading to altered localization and
stability, changes in the activity of the nuclear import and export machinery and protein-
protein interactions which could retain the protein in a compartment. Given the sophisticated
regulation of the PP2A enzyme complexes and their numerous targets, literature mining was
performed to obtain a plausible candidate mechanism. Of the cytosolic proteins promoting
tumor survival, the family of AKT kinases is of outstanding importance (31). AKT is
expressed in high abundance in tumors (32) and PP2A family members are responsible for
the dephosphorylation of AKT (33, 34) . Importantly, in a very different context it was
suggested that AKT can be oxidized which promotes its interaction with PP2A (35). On this
basis we speculate that the cytosolic interaction of PP2A and AKT could contribute to the
mechanism of the altered localization of PP2A.
If our working model was correct, nuclear translocation of PP2A in the absence of Nox4
should result in a decreased association of PP2A with its target AKT and a subsequent
increase in AKT phosphorylation, which was indeed the case (Fig. 3G&H). Importantly, we
found no evidence that the pathway leading to increased AKT phosphorylation, like the
formation of PIP3 or PTEN oxidation, expression and activity were responsible for the
increase in AKT phosphorylation (Supplemental figure 5&6).
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AKT is a redox-target of Nox4
In order to determine whether AKT could be the redox-switch responsible for PP2A
translocation, Redox-BIAM switch assays were performed, which indeed demonstrated that
loss of Nox4 resulted in a significant reduction of AKT oxidation (Fig. 4A). As targeted redox-
proteomics approach did not yield sufficient coverage of the peptide sequence of AKT
isolated out of tissues, the redox-active cysteines of AKT were mapped from the recombinant
protein. AKT was reduced and subsequently incubated with low amounts of H2O2. This
redox-stimuli resulted in disulfide bridge formation between cysteine 60 and 77 and between
cysteine 296 and 310, the latter being conserved in all AKT homologues (Fig. 4B &
Supplemental figure 7, Supplemental table 1&2). Based on these data it could be
speculated that Nox4-dependent H2O2 formation changes the confirmation of AKT by altering
disulfide bridge formation.
Oxidized AKT interacts with the PPP2R1A scaffolding subunit of PP2A
Next, the redox-nature of the interaction of PP2A and AKT was defined. Immunoprecipitation
of AKT followed by proteomics analysis revealed that PPP2R1A co-precipitates with AKT
(Fig. 4C, Supplemental table 3). PPP2R1A, also known as PP2A, subunit A, R1-α isoform
or PR65-α, is a scaffolding molecule that coordinates the assembly of the catalytic C subunit
and the variable regulatory B subunit. Unfortunately no B or C subunit of PP2A was
recovered in these screens. This might be consequence of the fact that the PPP2R1A
subunit of PP2A mediates the interaction with other proteins (36). Potentially, detergent
conditions were so stringent in order to gain specificity that the PP2A protein complex was
disrupted.
To substantiate our findings, an antibody directed against both C-subunit of PP2A was used
(37) and regular co-immunoprecipitation experiments followed by Western blot were
performed. These experiments demonstrated an interaction of PP2A subunit C with AKT in
WT cells, which was impaired in Nox4-/- cells (Fig. 4D) Overexpression of redox dead
mutants of AKT (C296S C310S) did not restore co-precipitation of AKT with PP2A in Nox4
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knockout cells, whereas after overexpression of a kinase-dead mutant (T308A/S473A) more
PP2A was co-precipitated with AKT in the knockout cells (Fig. 4D). In keeping with this, PLA
assays demonstrated that the redox-dead mutants exhibited an attenuated interaction with
AKT even in Nox4 expressing WT cells (Fig. 4E). Collectively, these data suggest that
interaction of PP2A with AKT occurs through the subunit PPP2R1A and requires Nox4-
dependent oxidation of AKT but not the kinase function of AKT.
Kinase- but not Redox-dead AKT restores normal cell function in Nox4-/- cells
Next it was tested whether cytosolic retention of PP2A in the cytosol of WT cells is
dependent on the oxidation site of AKT. Overexpression of any of the three wild-type AKT
homologues restored cytosolic retention of PP2A. Importantly, this effect was also observed
when kinase-dead versions of AKT but not the redox-indicative cysteine-mutant of AKT was
overexpressed (Fig. 5A&B). According to our model, restoration of cytosolic trapping of
PP2A should also restore DNA damage detection and thus phosphorylation of H2AX. This
was indeed the case (Supplemental figure 8). Overexpression of wild-type AKT restored
γH2AX in Nox4-/- cells to the level observed in WT cell. In contrast, overexpression of redox-
dead AKT failed to have this effect. This would suggest that also DNA-repair is dependent on
the oxidation of AKT resulting in PP2A trapping. To address this, comet assays were
performed after overexpression of the different AKT plasmids. Wild-type AKT reduced the
number of comets in Nox4-/- cells to the level of WT cells and similar effects were observed
in kinase-dead mutants of AKT (Fig. 5C&D). In contrast, none of the redox-dead versions of
AKT was able to affect the number of comets. Thus, retention of PP2A by Nox4-oxidized
AKT stabilized γH2AX in the nucleus, which results in DNA repair and reduced number of
comets.
Discussion
In the present study we observed that genetic deletion of the NADPH oxidase Nox4 resulted
in genomic instability, which was a consequence of attenuated DNA damage response due
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to an increased dephosphorylation of H2AX and attenuated accumulation of p53. Under
physiological conditions, Nox4 provides a basal tone of H2O2 which oxidizes AKT and
subsequently sequesters the phosphatase PP2A in the cytosol. Deletion of Nox4 and thus
attenuated formation of H2O2 results in reduced AKT, which does not bind PP2A. As
consequence, the phosphatase is transported into the nucleus and dephosphorylates
γH2AX. DNA damage accumulates and results in genomic instability and tumor progression.
It has been widely accepted that ROS are potential harmful chemicals which facilitate lipid
peroxidation und DNA damage. ROS are mediators of UV and X-ray radiation as well as
environmental toxins like arsenide and cigarette smoke. These kinds of damaging ROS are,
however, highly reactive, like those forms produced by Fenton or Haber-Weiss chemistry in
the presence of transition metals (38, 39). Despite this, redox-reactions are ubiquitous in an
oxygen respiring cell and a complex thiol-redox system has developed in aerobic organism to
cope with the oxidizing power of oxygen. Cells very well resist a fairly broad concentration
range of oxidants and low reactive ROS and in particular H2O2 have evolved as important
signaling molecules. Endogenous ROS generator systems, like the Nox family, utilize
antioxidant response systems to mediate signal transduction (40). Moreover, during evolution
systems have developed which are redox-sensitive and are transiently inactivated during
signaling to allow signal propagation.
Work from several labs including ours suggests that the NADPH oxidase Nox4 has a unique
function in this respect. Nox4 constitutively produces H2O2 and thereby provides a basal
oxidative tone to the cell. Through this Nox4 is protective (9) and maintains the activity of
antioxidant systems like Nrf2 (10, 41) and protein kinase A (42)(43). H2O2 produced by Nox4
contributes to differentiation (16) which is in accordance to the concept that during the
progression from stem cells towards differentiated cells, cellular ROS production is increased
(44). In fact, hypoxia and exquisitely low ROS level are characteristics of stem cells and stem
cells niches (44).
Previously, it was noted that deletion of Nox4 results in attenuated differentiation and
inflammatory activation which was in part mediated by attenuated eNOS expression, Nrf2
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activity (10, 45, 46) and JNK signaling (16). With the present work we establish AKT as an
additional redox-target of Nox4 important for malignant transformation. Nox4 promoted the
interaction of PP2A with AKT, which facilitated AKT dephosphorylation. Given that AKT
promotes survival and proliferation, this finding explains the increased proliferation after
deletion of Nox4, observed not only in this but also other studies (39).
As a second effector of Nox4, H2A phosphorylation which is an indicator of DNA damage,
was identified. In case of DNA double-strand breaks, H2A is phosphorylated by ATM at
Ser139 (47) and this event is a prerequisite to cellular response to DNA-damage (29). The
present observation of attenuated γH2AX in the absence of Nox4 is in line with a previous
report that antioxidants reduce the level of γH2AX in murine lung cancer tumors (3). It
appears that the binding of PP2A to the highly abundant AKT deprives other cellular
compartments from this phosphatase. After Nox4 knockout AKT is less oxidized, PP2A is
released from AKT and translocates into the nucleus, where it dephosphorylates γH2AX (28).
Phosphatases are well known redox-switches as several families, such as dual-specific
phosphatases, the lipid phosphatase PTEN and tyrosine phosphatases are inactivated by
H2O2. In fact, it has been shown that Nox1 inactivates PTEN (48) and inhibition of Nox1
induces apoptosis by attenuating AKT signaling in cancer cells (49). PTP-inactivation by
Nox4-derived H2O2 is certainly occurring under certain conditions and might promote survival
of some cancer cells (50, 51); the tonic activity of Nox4, however, in most cells should result
in an induction of the antioxidant defense to counteract this process (41, 46, 52).
PP2A, the phosphatase of concern in the present work, however does not contain a redox-
sensitive cysteine in the active center but a metal (53), and is therefore not redox-sensitive.
The variable intracellular distribution of PP2A and its effect on cell survival might explain the
conflicting data obtained with PP2A inhibitors in cancer. There is this long standing history of
okadaic acid and other PP2A inhibitors as carcinogens (54), but there is growing controversy
regarding the role of PP2A as tumor suppressor. Inactivation of PP2A triggers tumor-
selective cell death (55) and the PP2A inhibitors cantharidin or okadaic acid induce apoptosis
of pancreatic cancer cells through persistent phosphorylation of IKKα and a sustained
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activation of the NF-κB pathway (56). Subcellular localization of PP2A activity may be the
key to understand the controversial findings. In fact the B subunit determinates the function
of the PP2A complex and localization of the phosphatase activity. Out of the four recognized
subfamilies; it is plausible, that a B56 complex may prevent DNA damage repair, if not
retained in the cytosol, as a B56ϵ containing PP2A holoenzyme has been described to
dephosphorylate γH2Ax (57).
An unexpected result of the present study was that Nox4 deletion elevated AKT
phosphorylation. It is known since very long that H2O2, by inhibition of PTEN and activation of
tyrosine kinases increases AKT phosphorylation. Moreover, in several tumor cell lines, Nox4,
as a source of H2O2 was suggested to drive AKT phosphorylation (58–62). Nevertheless, in
other studies AKT was identified as an inducer of Nox4 expression (63–66). These studies,
however, were all performed in transformed stable tumor cell lines. Thus, genomic instability
or malignancy induction is not concerned but rather a general aspect of how H2O2 promotes
migration and proliferation. In line with this, any mechanism reducing H2O2, like inhibition of
mitochondria, Nox1, Nox2 or Nox4 usually leads to similar protective response in these
studies (50, 67, 68)
Several studies report increased expression of Nox4 in cancer (60, 69). This is not surprising
as Nox4 is induced by hypoxia and fibrotic response, which is common in tumors. Thus,
Nox4 induction is unlikely to be causal in those studies. Moreover, despite the fact that
commercial antibodies are not specific for Nox4 or are not even detecting the enzyme (70),
many of the publications identify Nox4 by its molecular weight in Western blots or immuno
histology (for example) (60, 71).
Although our work suggests that Nox4 maintains genomic stability, other studies linked Nox
enzymes to the opposite effect. p22phox down-regulation contributes to genomic instability in
FLT3 expressing leukemia cells (72) and H-Ras transformed cell lines (73). Due to the
selection of the individual cell line, an association of this function to a single Nox enzyme is
impossible, and also most any homologue has been associated with DNA damage (74).
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These variable results stress that tumor research on cell lines should be interpreted with
great caution and that the results frequently cannot be transferred to the in vivo situation.
Our study is not the first linking Nox4 to a rather positive protective function in malignant
disease. In hepatic cancer, the Nox4 promoter is silenced which appears to promote hepato
carcinogenesis in rats (75). This data is even supported in a study looking at Nox expression
in hepatocellular carcinoma (76). High Nox1 expression was associated with less favorable
outcome, where high Nox4 expression was beneficial (76, 77). Thus, low Nox4 might thereby
be a prerequisite for tumor progression. Recently this view has been supported by a more
descriptive study where Nox4 siRNA prevented liver cancer in a xenograft-model (78) and in
a study of epidermal growth factor receptor inhibitor induced autophagy in head and neck
cancer cells in a Nox4 dependent way (79).
Collectively, with the present work, Nox4 was established as an endogenous source of ROS
which maintains genomic stability. This work supports clinical data that antioxidants do not
protect against cancer initiation and may even be harmful under certain conditions. The
dogmatic view of ROS as bona fide harmful molecules should be queried.
Acknowledgment
We are grateful to Sabine Harenkamp, Maria Walter and Jana Meisterknecht for their
excellent technical support of our study. Furthermore, we thank Itamar Goren and Beate
Fisslthaler for providing AKT mutant plasmids.
Conflict of interest
The authors of this manuscript have no conflict of interest in the present study.
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Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (to
KS SCHR1241/1-1 and SFB815/TP1, TP8 &Z1) and the Fraunhofer Gesellschaft (graduate
school translational research innovation pharma, TRIP).
References
1. Lonn E et al. (2005) Effects of long-term vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial. JAMA 293:1338–1347.
2. Omenn GS et al. (1996) Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial. J Natl Cancer Inst 88:1550–1559.
3. Sayin VI et al. (2014) Antioxidants accelerate lung cancer progression in mice. Sci Transl Med 6:221ra15.
4. Sirokmany G, Donko A, Geiszt M (2016) Nox/Duox Family of NADPH Oxidases: Lessons from Knockout Mouse Models. Trends in pharmacological sciences 37:318–327.
5. Brandes RP, Weissmann N, Schröder K (2014) Nox family NADPH oxidases: Molecular mechanisms of activation. Free radical biology & medicine 76C:208–226.
6. Groeger G, Mackey AM, Pettigrew CA, Bhatt L, Cotter TG (2009) Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide. Journal of neurochemistry 109:1544–1554.
7. Jiang F, Liu G-S, Dusting GJ, Chan EC (2014) NADPH oxidase-dependent redox signaling in TGF-beta-mediated fibrotic responses. Redox biology 2:267–272.
8. Menegon S, Columbano A, Giordano S (2016) The Dual Roles of NRF2 in Cancer. Trends in molecular medicine 22:578–593.
9. Schröder K et al. (2012) Nox4 is a protective reactive oxygen species generating vascular NADPH oxidase. Circulation research 110:1217–1225.
10. Zhang M et al. (2010) NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis. Proc Natl Acad Sci U S A 107:18121–18126.
11. Craige SM et al. (2015) Endothelial NADPH oxidase 4 protects ApoE-/- mice from atherosclerotic lesions. Free radical biology & medicine 89:1–7.
12. Di Marco E et al. (2016) NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis. Free radical biology & medicine 97:556–567.
13. Gray SP et al. (2016) Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling. Arteriosclerosis, thrombosis, and vascular biology 36:295–307.
14. Clempus RE et al. (2007) Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol 27:42–48.
15. Schröder K, Wandzioch K, Helmcke I, Brandes RP (2009) Nox4 acts as a switch between differentiation and proliferation in preadipocytes. Arterioscler Thromb Vasc Biol 29:239–245.
16. Goettsch C et al. (2013) NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis. J Clin Invest 123:4731–4738.
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
17. Laleu B et al. (2010) First in class, potent, and orally bioavailable NADPH oxidase isoform 4 (Nox4) inhibitors for the treatment of idiopathic pulmonary fibrosis. J Med Chem 53:7715–7730.
18. Gavazzi G et al. (2007) NOX1 deficiency protects from aortic dissection in response to angiotensin II. Hypertension 50:189–196.
19. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
20. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461.
21. Rubsamen D et al. (2014) Inflammatory conditions induce IRES-dependent translation of cyp24a1. PloS one 9:e85314.
22. Clark J et al. (2011) Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry. Nat Methods 8:267–272.
23. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature protocols 2:1896–1906.
24. Gotze M et al. (2012) StavroX--a software for analyzing crosslinked products in protein interaction studies. Journal of the American Society for Mass Spectrometry 23:76–87.
25. Ashwell MA et al. (2012) Discovery and optimization of a series of 3-(3-phenyl-3H-imidazo4,5-bpyridin-2-yl)pyridin-2-amines: orally bioavailable, selective, and potent ATP-independent Akt inhibitors. Journal of medicinal chemistry 55:5291–5310.
26. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature biotechnology 26:1367–1372.
27. Tyanova S et al. (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nature methods 13:731–740.
28. Chowdhury D et al. (2005) gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol Cell 20:801–809.
29. Keogh M-C et al. (2006) A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Nature 439:497–501.
30. Cohen P, Klumpp S, Schelling DL (1989) An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS letters 250:596–600.
31. Amaravadi R, Thompson CB (2005) The survival kinases Akt and Pim as potential pharmacological targets. The Journal of clinical investigation 115:2618–2624.
32. Bellacosa A, Kumar CC, Di Cristofano A, Testa JR (2005) Activation of AKT kinases in cancer: implications for therapeutic targeting. Advances in cancer research 94:29–86.
33. Millward TA, Zolnierowicz S, Hemmings BA (1999) Regulation of protein kinase cascades by protein phosphatase 2A. Trends in biochemical sciences 24:186–191.
34. Kuo Y-C et al. (2008) Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. The Journal of biological chemistry 283:1882–1892.
35. Murata H et al. (2003) Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt. J Biol Chem 278:50226–50233.
36. Krauss G, ed (2014) Biochemistry of signal transduction and regulation (Wiley-VCH, Weinheim).
37. Kiely M, Kiely PA (2015) PP2A: The Wolf in Sheep's Clothing? Cancers 7:648–669.
38. Mello Filho, A C, Hoffmann ME, Meneghini R (1984) Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron. Biochem J 218:273–275.
39. Franke TF, Kaplan DR, Cantley LC (1997) PI3K: downstream AKTion blocks apoptosis. Cell 88:435–437.
40. Brandes RP, Weissmann N, Schröder K (2014) Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases. Journal of molecular and cellular cardiology 73:70–79.
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
41. Brewer AC et al. (2011) Nox4 regulates Nrf2 and glutathione redox in cardiomyocytes in vivo. Free radical biology & medicine 51:205–215.
42. Burgoyne JR et al. (2015) Deficient angiogenesis in redox-dead Cys17Ser PKARIalpha knock-in mice. Nature communications 6:7920.
43. Burgoyne JR, Mongue-Din H, Eaton P, Shah AM (2012) Redox signaling in cardiac physiology and pathology. Circulation research 111:1091–1106.
44. Ushio-Fukai M, Rehman J (2014) Redox and metabolic regulation of stem/progenitor cells and their niche. Antioxidants & redox signaling 21:1587–1590.
45. Goy C et al. (2014) The imbalanced redox status in senescent endothelial cells is due to dysregulated Thioredoxin-1 and NADPH oxidase 4. Mitochondria, Metabolic Regulation and the Biology of Aging 56:45–52.
46. Smyrnias I et al. (2014) Nicotinamide Adenine Dinucleotide Phosphate Oxidase-4-Dependent Upregulation of Nuclear Factor Erythroid-Derived 2-Like 2 Protects the Heart During Chronic Pressure Overload. Hypertension.
47. Bonner WM et al. (2008) GammaH2AX and cancer. Nature reviews. Cancer 8:957–967.
48. Cui W et al. (2011) NOX1/nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase promotes proliferation of stellate cells and aggravates liver fibrosis induced by bile duct ligation. Hepatology 54:949–958.
49. Ito K et al. (2016) Inhibition of Nox1 induces apoptosis by attenuating the AKT signaling pathway in oral squamous cell carcinoma cell lines. Oncology reports 36:2991–2998.
50. Jayavelu AK et al. (2016) NOX4-driven ROS formation mediates PTP inactivation and cell transformation in FLT3ITD-positive AML cells. Leukemia 30:473–483.
51. Mondol AS, Tonks NK, Kamata T (2014) Nox4 redox regulation of PTP1B contributes to the proliferation and migration of glioblastoma cells by modulating tyrosine phosphorylation of coronin-1C. Free radical biology & medicine 67:285–291.
52. Nlandu-Khodo S et al. (2016) NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury. Scientific reports 6:38598.
53. Guo F et al. (2014) Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone. Cell Res. 24:190–203.
54. Fujiki H, Suganuma M (1993) Tumor promotion by inhibitors of protein phosphatases 1 and 2A: the okadaic acid class of compounds. Adv Cancer Res 61:143–194.
55. Zimmerman R et al. (2012) PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing. Cell Death Dis 3:e291.
56. Li W et al. (2011) PP2A inhibitors induce apoptosis in pancreatic cancer cell line PANC-1 through persistent phosphorylation of IKKalpha and sustained activation of the NF-kappaB pathway. Cancer Lett 304:117–127.
57. Li X, Nan A, Xiao Y, Chen Y, Lai Y (2015) PP2A-B56 complex is involved in dephosphorylation of gamma-H2AX in the repair process of CPT-induced DNA double-strand breaks. Toxicology 331:57–65.
58. Tanaka M et al. (2015) Inhibition of NADPH oxidase 4 induces apoptosis in malignant mesothelioma: Role of reactive oxygen species. Oncology reports 34:1726–1732.
59. Zhao QD et al. (2015) NADPH oxidase 4 induces cardiac fibrosis and hypertrophy through activating Akt/mTOR and NFkappaB signaling pathways. Circulation 131:643–655.
60. Zhang C et al. (2014) NOX4 promotes non-small cell lung cancer cell proliferation and metastasis through positive feedback regulation of PI3K/Akt signaling. Oncotarget 5:4392–4405.
61. Jafari N et al. (2017) CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells. PloS one 12:e0170327.
62. Crosas-Molist E et al. (2016) The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination. Oncogene.
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
63. Liu Z-M, Tseng H-Y, Tsai H-W, Su F-C, Huang H-S (2015) Transforming growth factor beta-interacting factor-induced malignant progression of hepatocellular carcinoma cells depends on superoxide production from Nox4. Free radical biology & medicine 84:54–64.
64. Choi J-A, Jung YS, Kim JY, Kim HM, Lim IK (2016) Inhibition of breast cancer invasion by TIS21/BTG2/Pc3-Akt1-Sp1-Nox4 pathway targeting actin nucleators, mDia genes. Oncogene 35:83–93.
65. Edderkaoui M et al. (2013) NADPH oxidase activation in pancreatic cancer cells is mediated through Akt-dependent up-regulation of p22phox. The Journal of biological chemistry 288:36259.
66. Govindarajan B et al. (2007) Overexpression of Akt converts radial growth melanoma to vertical growth melanoma. The Journal of clinical investigation 117:719–729.
67. Zhang H-S, Sang W-W, Ruan Z, Wang Y-O (2011) Akt/Nox2/NF-kappaB signaling pathway is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation. Archives of biochemistry and biophysics 505:266–272.
68. Kodama R et al. (2013) ROS-generating oxidases Nox1 and Nox4 contribute to oncogenic Ras-induced premature senescence. Genes to cells : devoted to molecular & cellular mechanisms 18:32–41.
69. Roy K et al. (2015) NADPH oxidases and cancer. Clinical science (London, England : 1979) 128:863–875.
70. Babelova A et al. (2012) Role of Nox4 in murine models of kidney disease. Free radical biology & medicine 53:842–853.
71. Singh L et al. (2016) Prognostic significance of NADPH oxidase-4 as an indicator of reactive oxygen species stress in human retinoblastoma. International journal of clinical oncology 21:651–657.
72. Stanicka J, Russell EG, Woolley JF, Cotter TG (2015) NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells. The Journal of biological chemistry 290:9348–9361.
73. Weyemi U et al. (2012) ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence. Oncogene 31:1117–1129.
74. Kang MA, So E-Y, Simons AL, Spitz DR, Ouchi T (2012) DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway. Cell death & disease 3:e249.
75. Lopez-Alvarez GS et al. (2017) Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats. Biology open 6:59–70.
76. Lu C-L et al. (2011) NADPH oxidase DUOX1 and DUOX2 but not NOX4 are independent predictors in hepatocellular carcinoma after hepatectomy. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 32:1173–1182.
77. Ha SY et al. (2016) NADPH Oxidase 1 and NADPH Oxidase 4 Have Opposite Prognostic Effects for Patients with Hepatocellular Carcinoma after Hepatectomy. Gut and liver 10:826–835.
78. Crosas-Molist E et al. (2014) The NADPH oxidase NOX4 inhibits hepatocyte proliferation and liver cancer progression. Free radical biology & medicine 69:338–347.
79. Sobhakumari A et al. (2013) NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells. Toxicol Appl Pharmacol 272:736–745.
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
0
20
40
60
80 WT
Nox4-/-
Comet Assay
DN
A-f
ragm
enta
ion
[%]
0
5
10
60
70
80 WT
Nox4*/*
6 8 10 12 14 16 180
20
40
60
80
100
WT
Nox4-/-
6 8 10 12 14 16 180
20
40
60
80
100
WTNox4 -/-
Figure 1. Nox4 knockout promotes tumor development and induces genomic instability.(A-C) AOM-DSS colon carcinogenesis model: Representative macro photo (A), H&E overview (B) and average number of solidcarcinoma nodules (C) in the colon induced by AOM/DSS. (n=11), *p<0.05 (D-F): MCA fibro sarcoma model. Fibro sarcomadevelopment (D) and tumor size (E) (100% equals the maximal tumor size of 1.5 cm, n=10). (F) Relative tumor growth. (G&H).Sarcomas and colon carcinomas stained for phosphorylated histone H3 (pH3) as proliferation marker. The statistics indicate therelative staining intensity (n=5-9). (I&J) Measurement of DNA double-strand breaks in fibro sarcoma cells was performed usingComet Assay (n=5). Comet Assay of cells treated with or without H2O2 (24h, 5 µM). Original pictures (I) and statistics (J) *p<0.05;WT vs. Nox4-/-, #p<0.05 CTL vs. H2O2 treated. (K) Conditional tamoxifen-inducible knockout mice were treated with tamoxifen for10 day and one week later cells were isolated from multiple organs and analyzed for DNA-fragmentation by Nicoletti Assay (n=3).
WT Nox4-/-
A B*
Tum
ors
/ col
on
C
I
ED
Nox4-/- 0
2
4
6
8
p<0.05
WT
[%]
[% o
f 1,5
cm]
Weeks after MCA Weeks after MCA
Tumor sizeTumor free animals
0.0
0.5
1.0
1.5
Tumor growth
[%]
*
WT Nox4-/-
H2O2
*
WT Nox4-/-
F
p<0.05
KWT Nox4-/-
WT + H2O2 Nox4-/- + H2O2
Nicoletti Assay
Lung Kidney Gut
J
**
*
Com
ets
[%]
WT: 1.0±0.1% Nox4-/-: 2.4±0.4*WT: 1.0±0.2%Nox4-/- 2.8±0.3*
G H
CTL
Fibro sarcoma Phospho-Histone H3 Colon carcinoma Phospho-Histone H3
#
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
0.0
0.5
1.0
Sarcoma cells
*
WT Nox4-/- WT Nox4-/-
H2O2
0.0
0.5
1.0
0
1
2
3
4
0.0
0.5
1.0
5
10
Colon & carcinoma
*
*
#
#
γH2A
X/β
-Act
in
WT Nox4-/- WT Nox4-/-
healthy carcinoma
B D
*
C SarcomaWT: 1.0±0.4
Nox4-/- 0.2±0.04*
Figure 2. Nox4-deficiency results in an enhanced DNA instability.The expression of the DNA damage marker γH2AX was measured in healthy colon and colon carcinomas (n= 11 WT & 9 Nox4-/-)(A) and in fibro sarcoma tissue (B) (n=4) by western blot. (C) Cross-sections of the skin of WT and Nox4-/- mice stained forphosphorylated H2AX 20 days after MCA injection as a sign for DNA damage. (D) Western blot for γH2AX in primary skinfibroblasts treated with or without MCA for 3 days (5µg/ml, n=3). *p<0.05 WT vs. Nox4-/-,#p<0.05 WT/Nox4-/- healthy/CTL vs.WT/Nox4-/- carcinoma/MCA. (E-H) p53 abundance. mRNA (E&F) and protein (G&H) of p53 in fibro sarcoma tissue (E&G) andisolated fibro sarcoma cells (F&H). Numbers below the blots are the results of the densitometry. *p<0.05. n>5. H2O2 denotestreatment with 5µM for 24h. (I) p53 mRNA expression was measured by RT-qPCR after MCA treatment (1-5 days, 5µg/ml) in freshlyisolated lung endothelial cells (n=5)
*
Skin fibroblasts
#
γH2AX
β-Actin
MCAWT Nox4-/-
WT Nox4-/-
MCA
* #
A
E
0.0
0.5
1.0
*
Sarcoma
rel.
p53
mR
NA
exp
ress
ion
WT Nox4-/-
p53
β-Actin
Sarcoma cells
WT Nox4-/-
p53
β-Actin
0.2±.1*1±.2
0.3±.1*1±.2
SarcomaG
H
F Lung endothelial cells
1 2 3 4 50
5
10
15WTNox4-/-
days MCA
rel.
p53
mR
NA
p<0.05
I
CTL CTL
#
CTL
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
H2O2
Nox4-/-
1nM OANox4-/-
B
DC
E
A
1±.2
Nucleus Cytosol
WT Nox4-/- WT Nox4-/-
Topo-I/ β-Actin
PP2A
1.9±.3* 0.7±.1*1.1±.1
PP2A localization
0.0
0.1
0.2
0.3
WT N4-/-
Cytosol
*
Abs
orba
nce
@ 4
05nm
Phosphatase activity
0.00
0.02
0.04
0.06
0.08 *
WT N4-/-WT N4-/-
DMSO 1nM OA
Nucleus
DMSONox4-/-WT WT
1±.05 0.6±.1* 1.2±.4 1.6±.5#
γH2AX
β-Actin
CTL
Nox4-/-WT WT
γH2AX
β-Actin
1±.01 0.6±.02* 1.1±.2 1±.3 #
F Nuclear PP2A
CTL
Nox4-/-
H2O2
WT Nox4-/-WT1±.2 2.0±.25* 1.4±.4 0.9±.06
PP2A
TOPO-I
Blue: DAPIGreen: PLA PP2A & γH2AX
WT: 38.5±3.2% Nox4-/-: 51.3±3.8%*
Figure 3. Nox4-deficiency enhances nuclear PP2A activity, this decreases nuclear γH2AX but increases cytosolic AKTphosphorylation.(A) Interaction between PP2A and γH2AX as determined by proximity ligation assay. Quantification of co-localization relative to thenumber of cells stained with DAPI in %. (B) PP2A abundance in cytosolic and nucleus fraction as evaluated by western blot. (C)Serine-threonine phosphatase activity as measured under basal conditions and after treatment with okadaic acid (OA) in cytosoland nucleus with the aid of NPP as an artificial substrate . (D&E) γH2AX abundance as quantified by immunoblotting with andwithout 1nM of the PP2A inhibitor okadaic acid (OA) overnight (D) or H2O2 (24h, 5 µM, E). (F) Nuclear expression of PP2A aftertreatment with or without H2O2 as determined by western blot. All experiments were carried out in isolated fibrosarcoma cells (F).Statistics are represented as numbers below the representative western blots; *p<0.05 WT vs. Nox4-/-,#p<0.05 Nox4-/- CTL vs.Nox4-/- OA/H2O2, n=5. (G) Interaction between AKT and PP2A as determined by proximity ligation assay. Quantification of co-localization relative to the number of cells stained with DAPI in % (H). Western blot for phosphorylation of AKT at Ser473 in fibrosarcoma tissue, colon carcinoma tissue and isolated fibro sarcoma cells of WT and Nox4 -/- mice.
G
1.4±.1*1±.16
p-AKT
AKT
WT Nox4-/-2±.5*1±.05
Fibro sarcoma
Colon carcinoma
1.3±.21±.12
Sarcomacells
H
WT Nox4-/-WT Nox4-/-
WT: 56.5±5% Nox4-/- : 35.6±3%*
Blue: DAPIGreen: PLA PP2A & AKT
PP2A total
β-Actin
PP2A
1.04±.031±.04
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
Figure 4. The interaction of PP2A and AKT is redox-sensitive.(A) AKT redox modification as analyzed by BIAM Switch Assay in fibro sarcoma cells of wildtype and Nox4-/- (n=3). (B) Mappingof the H2O2-dependently formed disulfide bridges on the structure of human AKT1 as obtained from 4EJN [25] (seeSupplementary table 1&2. (C) AKT1-3 proteins were immune-captured and co-purified interacting proteins were identified byLC/MS. Lable free quantification (LFQ) values were statistical analyzed using permutation test (FDR <0.05, 250 randomizations),see supplementary table 3. Blue and red dots represent significant enriched proteins. Black and grey dots were enriched innegative control or background, respectively. Marked protein Ppp2r1a was found to be significant enriched in AKT1-3 pull-downsamples. (D) Co-immunoprecipitation of AKT followed by western blotting for PP2A catalytic subunit after overexpression withGFP, the redox-dead mutants for AKT1-3 and phospho-dead AKT1 mutant.(E) Proximity ligation in wildtype fibro sarcoma cells after overexpression of AKT1, AKT3, AKT1 and AKT3 redox-dead mutants.Antibodies used for AKT1 were PP2A and HA and for AKT3 PP2A and GFP. Results of cells overexpressed with wildtype AKTwere set to 100%.
A
0.0
0.5
1.0
WT Nox4-/-
IP:BIAM
Ini
Post-IP
*
BIAM Switch AssayWB AKT
Oxi
dize
d A
KT
CB
D
Nox4-/-WT
IniIB: AKT
IP: AKTIB: AKT
Nox4-/-WT
IP: AKTIB: PP2A
Redox-dead AKT-GFP
Kinase-dead AKT-FlagPlasmid GFP
Nox4-/-WT
AKT1: 100±5.6%
AKT1 C296S/C310S : 86.5±5%
AKT3: 100±5.5%
AKT3 C293S/C307S: 81.5±5.5%
E
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
Figure 5: PP2A translocation can be rescued by overexpressing AKT.PP2A (catalytic subunit) localization in the cytosol (A) and nucleus (B) was determined by western blot after nuclear extraction.Cells were overexpressed with the plasmids indicated. *p<0.05; WT/WT mutated AKT vs. Nox4-/-/Nox4-/- mutated AKT, #p<0.05Nox4-/- vs. Nox4-/- AKT OE, n=5 ) (C) Comet Assay , Exemplary photos (C) and statistics (D) of WT and Nox4-/- cells withoverexpression of the plasmids indicated *p<0.05 WT vs Nox4-/-, #p<0.05 Nox4 GFP vs. Nox4-/- AKT mutant. n=5
0.0
0.5
1.0
1.5WT
N4-/-
A PP2A Cytosol
β-Tubulin
PP2A
*
# #
*
GFP AKT1 AKT3 AKTCys-Mut
AKT2
PP
2A/β
-Tub
ulin
PP2A NucleusB
PP
2A/T
OP
O-I
#
0
1
2
3WT
N4-/-*
##
*
**
GFP TASA GFP K179M
*#
TOPO-I
PP2A
GFP AKT1 AKT3 AKTCys-Mut
AKT2 GFP TASA GFP K179M
0
20
40
60
WTNox4-/-
GFP AKT1 AKT2 AKT3 AKT1C296/310S
AKT3C293/307S
AKT2C297/311S
AKT1-3
Cys-Mut
AKTT308A/S473A
AKTK179M
* ** * *
# ## # #
C WT
Nox4-/-
+AKT1-3 CysMut+AKT1 +AKT K179M
D
Nox4-/-
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
5 6 7 8 9 10 11 12 13 14 15 16 17 180
50
100
150 WTNox1y/-
5 6 7 8 9 10 11 12 13 14 15 16 17 180
20
40
60
80 WTNox1y/-
**
**
*
*
Supplemental figure 1: Nox1-deficiency attenuates tumor development whereas the knockout of Nox2 has no effect ontumor development.WT littermates and corresponding Nox1y/- and Nox2y/- mice were injected subcutaneously with MCA. Fibro sarcoma developmentafter MCA-injection in Nox1y/- (A) and Nox2y/- mice (B) and tumor diameter in % (100% is equal to 1.5 cm) as assessed weeklywith a caliper in WT littermates, Nox1y/- (C) and Nox2y/- (D). *p<0.05 WT vs. Nox1y/- , n=10
A B
C D
5 6 7 8 9 10 11 12 13 14 15 16 17 180
50
100
150 WTNox2y/-
5 6 7 8 9 10 11 12 13 14 15 16 17 180
10
20
30
40
50 WTNox2y/-
Sar
com
a-fr
ee m
ice
[%]
Sar
com
a-fr
ee m
ice
[%]
Tum
or s
ize
[%]
Tum
or s
ize
[%]
*
Weeks after MCA-injection Weeks after MCA-injection
Weeks after MCA-injection Weeks after MCA-injection
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
Sar
com
a-fr
ee m
ice
[%]
Sar
com
a-fr
ee m
ice
[%]
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200
50
100
150 WTConditional endothelial specificNox4 knockout
12 13 14 15 16 17 18 19 200
50
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150 WTMacrophage-specific Nox4 knockout
A
B
C
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200
50
100
150 WTConditional systemic Nox4 knockout
Sar
com
a-fr
ee m
ice
[%]
Weeks after MCA-injection
Supplemental figure 2: Fibro sarcoma development in response to MCA in conditional Nox4 knockout.(A) Nox4flox/flox-CMV-CreERT2+/0 vs. Nox4flox/flox , all after tamoxifen-treatment (B) Conditional endothelial-specific (Nox4flox/flox-cdh5-CreERT2+/0 vs. Nox4flox/flox) all after tamoxifen-treatment. (C) LysM-macrophage and LysM neutrophil specific (Nox4flox/flox-LysM-Cre+/0 vs. Nox4flox/flox). Fibro sarcoma development was assessed weekly with a caliper. Mice were sacrificed when tumorreached 1.5 cm, n=4-10
Weeks after MCA-injection
Weeks after MCA-injection
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
pp53
p53
pp53/ p531.2±.31±.1
WT Nox4-/-
Nucleus
pp53
p53
pp53/ p531.1±.21±.04
WT Nox4-/-
Total
NQO1
β-Actin
NQO1protein expressionE
WT Nox4-/-0.8±.11±.1 NQO1/ β-Actin
A
B
0.5 1.0
WT
Nox4-/-
rel. MDM2 mRNA expression
*
Supplemental figure 3: p53 phosphorylation and translation is similar between WT and Nox4-deficient fibrosarcoma cells.(A&B) Western blot for p53 and phospho-p53 from nuclear (A) and total cellular extracts (B) of fibro sarcoma cells.Numbers below the blot indicate the result of the densitometry. (C&D) Polysome analysis was performed for investigationof translation. Absorbance was measured at 254 nm after polysomal fractionation in WT and Nox4-/- fibro sarcoma cells(C). Fractions were collected and analyzed for p53 mRNA expression with RT-qPCR. Normalization was performed tohousekeeping gene GAPDH (D). n=3; (E&F): NQO1 expression in fibro sarcoma cells was measured by Western blot (E).MDM2 (F) gene expression as quantified by RT-qPCR. *p<0.05
C
D
Monosomes Polysomes
-1.0
-0.5
0.0
0.5
1.0
λ=25
4nm
pol
ysom
alfr
actio
natio
nof
N
ox4-
/-vs
. WT
Polysome profile of wildtype and Nox4-/-fibro sarcoma cells
Polysome bound p53 in Nox4-/- vs. Wildtypefibro sarcoma cells
rela
tive
diffe
renc
ein
rib
osom
albi
ndin
gN
ox4-
/-vs
. WT
0.06
0.07
0.08
0.09
0.06
0.07
0.08
0.09
WildtypeNox4-/-
1 2 3 4 5 6 7 8 9 10
40S60S
80S
F
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
Supplemental figure 4: The kinase ATM is not differentially expressed or phosphorylated in Nox4-/-.(A) RT-qPCR was performed in WT and Nox4-/- fibro sarcoma cells after DMSO or MCA treatment for 3 days (5µg/ml). (B) ATMexpression was quantified by western blot. (C) As a readout for its activity the amount of phosphorylated ATM was measured bywestern blot and normalized to total ATM protein expression. n=6-7
0.0
0.5
1.0
0.0
0.5
1.0
p-A
TM
/AT
M
WT Nox4-/-
p-ATM
ATM
AT
M/T
OP
O-1
WT Nox4-/-
ATM
TOPO-I
ATM mRNA
0.0
0.5
1.0
1.5
rel.
mR
NA
expr
essi
on
ATM activityATM
WT N4-/-WT N4-/-
MCADMSO
A B C
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
0
2
4
6
WT Nox4-/-
PIP3/PI-Standard
0
1
2
WT Nox4-/-
PIP3/PIP2
0
2
4
6
8
10
C38:4 PIP3/PI-Standard
0
1
2
3
4
C38:4 PIP3/PIP2
Tumor Fibro sarcoma cells
Rat
io P
IP3/
PIP
2 [x
10-3
]
Rat
io P
IP3/
PI-
Sta
ndar
d [x
10-3
]
WT N4-/-
CTL
WT N4-/-
TNFα
Rat
io P
IP3/
PI-
Sta
ndar
d [x
10-3
]
Rat
io P
IP3/
PIP
2 [x
10-3
]
WT N4-/-
CTL
WT N4-/-
TNFα
B C
p85
β-Actin
p110
WT Nox4-/-
1.2±.1*1±0
1.3±.41±.2
A
p85/β-Actin
p110/β-Actin
Supplemental figure 5: PI3-Kinase activity is unchanged even if expression is slightly increased in the absence ofNox4.Protein abundance (A) of the two subunits of the PI3 Kinase, p85 and p110, was assessed by western blot. LC-MS/MSmeasurements for PIP3 and PIP2 in fibro sarcoma tissue (B) and isolated fibro sarcoma cells with or w/o TNFα (10ng/ml) (C).Data expressed as C18:0 C20:4 PIP3 response ratio normalized to C18:0 C20:4 PI response ratio to account for cell inputvariation. Statistics are represented as numbers below the representative western blots; *p<0.05, n=4
Supplemental figure 6: Nox4 knockout in fibro sarcoma cells does not increase the level of reduced PTEN.(A) Total PTEN expression was assessed by western blot. Reduced (B) vs. oxidized (C) PTEN as evaluated under non-reducing conditions by western blot. n=5
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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
Supplemental figure 7: Cys60/77 and Cys296/310 are redox-sensitive and form disulfidesRecombinant human AKT1 (Uniprot ID P31749) was reduced and treated with H2O2. Samples were digested withtrypsin and analyzed by LC/MS. (A) The following thiol oxidations on cysteines were identified by proteomics softwarePeaks7.0: sulfinic acid (Dioxidation), sulfonic acid (Trioxidation) and loss of the mass of one (-1) or two (-2) hydrogensrefer to formation of a disulfide bridge. For a list of all identified peptides, see supplementary table 1. (B) A specialsoftware to identify crosslinked peptides (StavroX, [24]) was further used to identify disulfides. Among the top-scoredcandidates for disulfides were Cys60/77 and Cys296/310 (Supplementary table 2). Figure shows position of fragmentions within a di-peptide.
.
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
0.0
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Supplemental Figure 8. Nox4-dependent oxidation of AKT is necessary for γH2AXphosphorylation and prevention of DNA damage.γH2AX expression determined by western blot after overexpression of the plasmids indicated(n=5); *p<0.05; WT/WT mutated AKT vs. Nox4-/-/Nox4-/- mutated AKT, #p<0.05 Nox4-/- vs.Nox4-/- AKT OE, n=5
γH2AX after AKT overexpression
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GFP AKT1 AKT3 AKT1-3 Mut
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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 16, 2017. ; https://doi.org/10.1101/177055doi: bioRxiv preprint
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